Optimization and scale up of the Baeyer-Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHL) was studied in order to demonstrate this technology on 100 L pilot plant scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) that utilizes the costly redox cofactor NADPH, which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell free extract, whole cells, fermentation broth and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated resulting in similar performances. Substrate dosing rate and biocatalyst loadings were optimized. At 30 mL scale, the best conditions were found when 30 mM h-1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied at 1 liter scale with 92% conversion, achieving a specific activity of 13.3 U g-1 cell wet weight (cww), a space time yield of 3.4 g CHL L-1 h-1 and a biocatalyst yield of 0.83 g CHL g-1 cww. A final 100 liter demonstration was performed in a pilot plant facility. After 9 hours, the reaction reached 85% conversion, 12.8 U g-1 cww, a space time yield of 2.7 g L-1 h-1 and a biocatalyst yield of 0.60 g CHL g-1 cww. The extraction of product resulted in 2.58 kg isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).
Alcohol dehydrogenases (ADH) are widely used to enantioselectively reduce ketones to chiral alcohols, but their application in industrial scale oxidations is rare. Reasons are the need for an NAD(P)+ cofactor regeneration system, often low performance in oxidative reactions and the limited substrate scope of ADHs. ADHA from Candida magnoliae DSMZ 70638 is identified to efficiently catalyze the regio‐selective hydroxy‐lactone oxidations to hydroxy‐lactones. Hydroxy‐lactones are common intermediates in industrial processes to cholesterol lowering (va)statin drugs. A biocatalytic aliphatic hydroxy‐lactone oxidation process is developed using pure oxygen as oxidant reaching volumetric productivities of up to 12 g L−1 h−1, product concentrations of almost 50 g L−1 and 95% reaction yield. For co‐factor recycling a previously engineered, water‐forming NAD(P)H‐oxidase from Streptococcus mutans is used. The process is scaled up to industrial pilot plant scale and it could be demonstrated that ADH catalyzed oxidations can be developed to efficient and safe processes. However, the ADHA wild‐type enzyme is not productive enough in chlorolactol oxidation. Therefore, enzyme engineering and multi‐parameter screening is successfully applied to optimize the enzyme for the target reaction. The optimized ADHA variant shows a 17‐fold higher oxidative activity, a 26°C increased stability and is applied to develop an efficient chlorolactol oxidation process.
Alcohol dehydrogenases are able to catalyze the conversion of alcohols to aldehydes or ketones, simultaneously reducing the cofactor NAD + or NADP + to NAD(P)H. Because of the high costs of these pyridine cofactors, in situ cofactor regeneration is required for preparative applications in order to reach turnover numbers that are sufficient for economically viable processes. Here we present the development of a process for the enantioselective oxidation of rac-1-phenylethanol to acetophenone, applying an alcohol dehydrogenase coupled with an NAD(P)H oxidase for the enzymatic cofactor regeneration, which is active towards NADH as well as NADPH. The reaction system was investigated in view of various influential parameters with main focus on the external oxygen supply. We could show that a gassed stirred tank reactor is a promising reactor concept to run NAD(P)H oxidase-coupled alcohol dehydrogenase oxidations, including the possibility to scale-up the system. ■ INTRODUCTIONOxidations are a powerful synthetic tool, which is abundantly applied in academic research. Nevertheless their use for the pharmaceutical and fine chemical industry at a preparative scale is still limited due to several characteristics. Conventional chemical oxidations exhibit a lack of selectivity, hence requiring additional protecting groups to avoid the risk of overoxidation. Moreover, chemical oxidation processes are usually operated under high temperature and pressure. The reactions often lead to undesired byproducts, which can be toxic or smelly, making further downstream processing inevitable. Additionally, potential safety issues exist that have to be fulfilled on a large scale. In consequence a demand for greener, more efficient catalytic solutions persists, including regio-, stereo-, and enantioselective methods. Biocatalytic oxidations have the potential to fulfill these requirements. Applied biocatalystseither crude preparations or isolated enzymesare typically characterized by high selectivities preventing the need of further protection groups and therefore driving the oxidations more atomefficient. Moreover, enzymes are active under mild process conditions. These properties can lead to new process routes which reduce production costs and simultaneously account for a lower environmental pollution. 1,2 Alcohol dehydrogenases (ADHs) belong to the enzyme class of oxidoreductases and facilitate the interconversion between alcohols and aldehydes or ketones, requiring the supply of NAD or NADP, while most ADHs are strictly specific for one of these nicotinamide cofactors. For an effectively running process the stoichiometric use of the cofactors is unfeasible due to their costs. Total turnover numbers of 10 3 to 10 5 may be sufficient for an economically viable process, depending on the value of the reaction product. 3 Therefore, the cofactor has to be regenerated in situ. This challenge can be approached in many different ways. 4,5 For the regeneration of NAD(P) + , an enzymatic approach is presently the most profitable method, leading ...
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